Rapid-acting antidepressant drugs modulate affective bias in rats

How rapid-acting antidepressants (RAADs), such as ketamine, induce immediate and sustained improvements in mood in patients with major depressive disorder (MDD) is poorly understood. A core feature of MDD is the prevalence of cognitive processing biases associated with negative affective states, and the alleviation of negative affective biases may be an index of response to drug treatment. Here, we used an affective bias behavioral test in rats, based on an associative learning task, to investigate the effects of RAADs. To generate an affective bias, animals learned to associate two different digging substrates with a food reward in the presence or absence of an affective state manipulation. A choice between the two reward-associated digging substrates was used to quantify the affective bias generated. Acute treatment with the RAADs ketamine, scopolamine or psilocybin selectively attenuated a negative affective bias in the affective bias test. Low, but not high, doses of ketamine and psilocybin reversed the valence of the negative affective bias 24 hours after RAAD treatment. Only psilocybin, but not ketamine or scopolamine, led to a positive affective bias that was dependent on new learning and memory formation. The re-learning effects of ketamine were dependent on protein synthesis localised to the rat medial prefrontal cortex and could be modulated by cue-reactivation, consistent with experience-dependent neural plasticity. These findings suggest a neuropsychological mechanism that may explain both the acute and sustained effects of RAADs, potentially linking their effects on neural plasticity with affective bias modulation in a rodent model.


Animals and housing
All rats weighed between 300-350g at the start of training (12-13 Weeks of age).The sample size was based on our previous affective bias test (ABT) studies and a meta-analysis which suggested a medium to large effect size for the drug-induced negative bias and rewardinduced bias in Lister Hooded rats (13,14).All animals were pair-housed in standard enriched laboratory cages (55x35x21cm) with woodchip, paper bedding, cotton rope, wood chew, cardboard tube and red Perspex house (30x17x10cm), under a 12:12h reverse light-dark cycle (lights off at 08:00h) and in temperature-controlled conditions (21±1°C).The behavioural procedures and testing were performed during the animals' active phase between 09:00h and 17:00h.

Affective Bias Test (ABT) General protocol
Training: The ABT testing was carried out in a Perspex® arena (40x40cm) with two ceramic bowls (Ø 10cm) and a trio of digging substrates (reward-paired substrates -'A' or 'B' versus unrewarded substrate -'C', matched for digging effort and counterbalanced across subjects; for details, see Supplementary Table S1).Prior to ABT training animals underwent a five day habituation to handling with positive reinforcement (reward pellets) and two habituation sessions to the ABT arena (first without bowls, substrate or reward and second with empty bowls); rats were individually placed into the arena and allowed to explore for 10min.Further training consisted of three digging training sessions (20 trials per session) with a bowl filled with increasing amounts of digging substrate (sawdust) and a food reward (45mg purified rodent tablets, Test Diet, Sandown Scientific, UK).On the first day of digging training, each rat was placed in the arena and given 30s to approach and explore the empty bowl (without substrate) containing two pellets per trial.When the pellets were found and consumed, the trial was completed, and the rat was removed from the arena and the pellets were replenished in the bowl.During the next digging training session, each rat was given 30s to explore the bowl and start digging for a single pellet buried within 1 cm of sawdust.Following 20 trials in which the pellet was found and eaten, each rat was moved onto the final training session in which a single pellet was buried within 2 cm of sawdust.Once each animal was able to find a pellet within 30s on 10 consecutive trials (within a maximum 20 trials), the digging training was complete.
Following the training sessions, animals underwent a discrimination session allowing them to explore two bowls with two novel digging substrates (reward-paired substrate with single pellet versus unrewarded substrate).On each trial, the animal was individually placed in front of the two bowls.Once the animal made a choice by starting to dig in one bowl, the other bowl was removed by the experimenter.An example of a single discrimination trial is illustrated in supplementary movie S1.Choice of the reward-paired substrate was marked as a 'correct' trial, digging in the unrewarded substrate was classified as an 'incorrect' trial and if an animal failed to approach and explore the bowls within 30s, the trial was recorded as an 'omission'.Trials were continued until the rat achieved six consecutive correct choices for the rewardpaired substrate.The discrimination session allowed us to confirm that the animals could achieve our learning criterion of six consecutive correct trials in less than 20 trials.Once animals successfully reached criteria in the discrimination session, they were considered trained and progressed to testing in the reward learning assay.As detailed below, this test was carried out over 5 days and used to check that the cohort was correctly performing the task and at population level reward-induced positive bias was observed before animals progressed to studies involving affective state-induced biases and their modulation by RAADs.
Testing: Each week was composed of four pairing sessions (one per day) to generate two independent cue-specific memories (Supplemental figure S1).During the pairing sessions, each trial involved presenting the rat with a choice between two bowls containing two different digging substrates, one of which was reward-paired (substrate 'A' or 'B', counter-balanced across subjects and manipulation) and contained a single 45mg reward pellet, and the other of which was unrewarded (substrate 'C').Substrate C was kept the same for all four pairing sessions and a reward pellet was crushed into the bowl and mixed within the substrate, to prevent choices based on odour.One of substrates 'A' or 'B' was presented during pairing sessions on days 1 and 3, and the other was presented on days 2 and 4, with order counterbalanced across subjects (see Supplemental tables S2A-C).

Medial prefrontal cortex cannulation procedure
The surgical procedures were performed under inhalation anaesthesia of the isoflurane/O2 mix.The cannula was fixed to the skull with gentamicin bone cement (DePuy CMW, Johnson & Johnson, UK) and three stainless-steel screws.To reduce risks of infection and any blockage inside pins of the guide cannulae, dummy cannulae (Plastics One, UK) were placed inside and metal head caps secured on top.Animals received local anaesthetic during the surgery and following surgery were housed individually for ~3h and then allowed to fully recover for 11-14 days in pairs with free access to food and water.Postoperatively all animals were pair-housed in Techniplast high top cages (40.5 x 37.5 x 31 cm) with woodchip, paper bedding, cardboard tubes, wood chew and red Perspex houses (30 x 17 x 10 cm).

Infusion Procedure
The first habituation session involved animals being gently restrained, the dummy cannula removed, cleaned and then placed back.The second session involved dummy removal followed by insertion of the bilateral injection cannula (injector, 33-gauge, Plastics One, UK) extending 2.5mm beyond the length of the guide cannula into the mPFC and left in position for 5 min.without infusion.The injector was then removed, and the dummy cannula and head cap replaced.

Data analysis
For the memory retrieval studies involving a FG7142 or corticosterone-induced negative bias, animals which did not exhibit the expected negative bias under vehicle treatment were excluded.Applying this exclusion criteria led to the removal of four animals, one from the retrieval at 1h study with psilocybin (0.1-0.3mg/kg) treatment, three from the retrieval at 1h study with ketamine-anisomycin infusion and one from the retrieval at 24hrs study with ketamine infusion.We have also excluded one outlier (more than 2SD) from the ketamine new learning study, and animals that completed less than 15 trials during choice test, three animals from the reward learning assay with ketamine (25mg/kg) treatment, and one animal from the ketamine (1.0-25.0mg/kg)retrieval at 24hrs study.Their data was removed for all treatments in that study for choice bias data.

Supplementary Figures
Figure S1: The conceptual framework for the affective bias test (ABT) is outlined in panel A. The task builds from the observation that patient with major depressive disorder attribute less value to the memory of positive experiences indicative of a negative affective bias in learning and memory.The ABT was designed to enable the direct quantification of these affective biases in non-human species by generating two independent memories learnt under either an affective state manipulation or neutral/control state.Based on the concept that the arising affective bias will change the relative value of the reinforcer; this can be directly quantified by measuring the relative preference for the experience learnt during the affective state manipulation versus the neutral state.This concept was translated into a task which uses associative learning where animals learn to associate a specific digging substrate (examples in panel B) with a food reward.The value of each, independent learnt experience, is kept the same with each animal undergoing four paring sessions over 4 days followed by a choice test as illustrated in panel C.During the choice test, the two previously rewarded substrates are presented together for the first time and the animals preference quantified over 30 randomly reinforced trials.By administering an affective state manipulation or test treatment prior to one of the substrate-reward pairing sessions the affective bias generated during learning can be quantified at retrieval using a choice test.Extensive pharmacological and psychosocial manipulations confirmed the predictions illustrated in panel C.The ability of a treatment to induce an affective bias is tested by administering the test substance before the pairing sessions.The ability of a treatment to attenuate a negative affective bias already generated is tested by first inducing a negative affective bias using an established negative state induction method e.g.acute corticosterone or the benzodiazepine inverse agonist, FG7142, the administering the treatment either acutely or 24hrs before the choice test.To reduce the potential for carry over effects from the high dose of psilocybin and based on the data from the new learning protocol where an aversive effects was seen with 1mg/kg, the high dose was tested after the initial dose-response with a vehicle control group and fully counter-balanced.Although 1hr post treatment the negative bias was no longer evident in the one sample t-test, there was no significant difference between the vehicle and psilocybin group (panel A).The effects of 1mg/kg at 24hrs were also different from what was seen at the lower doses and although there was an attenuation of the negative bias, the inversion to a positive bias was not observed.Data shown as mean % choice bias ± SEM (bars) and individual data points (symbols), one sample t-test against a null hypothesised mean of 0% choice bias (***p<0.001) and pairwise comparisons using paired t-test ( ### p<0.001).The protocol was similar to the method used to investigate the sustained effects of RAADs on a negative affective bias but included a cue-reactivation step on day 5, 1 hour after administration of ketamine (1mg/kg).Animals were treated with either FG7142 (3mg/kg) or corticosterone (10mg/kg) to induce a negatively biased memory and then re-exposed to either the FG7142-paired cue or the vehicle paired cue for 3 secs before being returned to their home cage.Affective biases were quantified using a choice test 24 hours after ketamine and cue-reactivation.

Figure S9: No evidence of a recency effect of the last pairing session during the choice test of the cue-reactivation study.
In order to check whether the treatment-substrate-reward association learnt during the last pairing session had any impact on the memory retrieval in the cue re-activation study, we re-analysed the data based on whether the animal's last pairing session was the vehicle (Veh) or FG7142 (FG) treatment.Although the sample size for this analysis was reduced due to the counter-balanced design, there was no evidence that the last substrate-reward pairing session had any effects on the bias observed.The only group where there was a numerical difference observed was the ketamine (Ket) control re-exposure, mainly driven by a single value.Data shown as mean % choice bias ± SEM (bars) and individual data points (symbols, N=12 per treatment and 6 per condition), one sample t-test against a null hypothesised mean of 0% choice bias (*p<0.05,**p<0.01)and paired t-test with value adjusted for the number of comparisons.We propose that ketamine, psilocybin and scopolamine act in mPFC to alter glutamate signalling and shift E/I balance generating a 'therapeutic window' where emotional circuits are selectively disengaged or reset to their default mode (PHASE I).During the arising 'therapeutic window', retrieval of memories can occur in the absence of their associated affective bias and, under appropriate conditions, can be re-activated and re-learnt with a relatively more positive affective bias (PHASE II).These findings and this arising hypothesis may provide the missing link between preclinical studies suggesting neuroplastic effects and the rapid and sustained improvements in mood observed in the clinic.S2B : Standard procedure for testing in the reward learning assay.Each animal receives 2 pellets or 1 pellet counterbalanced over the four substrate-reward pairing sessions.Substrate (reward-paired substrates -'A' or 'B' versus unrewarded substrate -'C') and day are also counter-balanced resulting in four different groups.Table S9.Choice test data: number of head twitches and wet dog shakes following vehicle and drug treatments in the reward learning assay utilising psilocybin 0.1-1.0mg/kg.All drugs were administered 60 min.prior to the choice test.Animals were observed for 15 min.during testing session and the number of head twitches and wet dog shakes were scored.

Supplementary Tables
Table S12: Pairing sessions data: number of head twitches and wet dog shakes following vehicle and drug treatments in the new learning study.All drugs were administered 60 min.prior to substrate-reward pairing session (PS).Animals were observed for 10 min.during pairing session and the number of head twitches and wet dog shakes were scored.

Figure S2 :
Figure S2: Overview of the affective bias test protocol used to investigate the acute effects of RAADs on a negative affective bias.Animals were treated with either FG7142 (3mg/kg) or corticosterone (10mg/kg) to induce a negatively biased memory.Affective biases were quantified using a choice test with the RAAD or vehicle administered 1 hour before testing to investigate the acute effects on retrieval.

Figure S3 :
Figure S3: Effects of high dose psilocybin (1.0mg/kg) on acute (panel A) and sustained (panel B) modulation of a negative affective biases.To reduce the potential for carry over effects from the high dose of psilocybin and based on the data from the new learning protocol where an aversive effects was seen with 1mg/kg, the high dose was tested after the initial dose-response with a vehicle control group and fully counter-balanced.Although 1hr post treatment the negative bias was no longer evident in the one sample t-test, there was no significant difference between the vehicle and psilocybin group (panel A).The effects of 1mg/kg at 24hrs were also different from what was seen at the lower doses and although there was an attenuation of the negative bias, the inversion to a positive bias was not observed.Data shown as mean % choice bias ± SEM (bars) and individual data points (symbols), one sample t-test against a null hypothesised mean of 0% choice bias (***p<0.001) and pairwise comparisons using paired t-test ( ### p<0.001).

Figure S4 :
Figure S4: Effects of mid (10mg/kg) and high (25mg/kg) doses of ketamine on omissions (panel A) and latency (panel B) during the choice test.The choice test was terminated if animals had 10 or more omissions and although the choice data for the 25mg/kg dose is included for all subjects, only 2 animals completed the full 30 trials.Data shown as mean % choice bias ± SEM (bars) and individual data points (symbols), pairwise comparisons using paired t-test ( ## p<0.01, ### p<0.001).

Figure S5 :
Figure S5:Overview of the reward learning assay protocol used to investigate the acute effects of RAADs on a reward-induced bias.Animals were in the same affective state throughout training and testing but learnt to associate one of the substrate-reward cues with a higher value reward (2 reward pellets) or a low value reward (1 reward pellet).The reward -induced bias was quantified using a choice test with the RAAD or vehicle administered 1 hour before testing to investigate the acute effects on retrieval.

Figure S6 :
Figure S6:Overview of the affective bias test protocol used to investigate the sustained effects of RAADs on a negative affective bias.Animals were treated with either FG7142 (3mg/kg) or corticosterone (10mg/kg) to induce a negatively biased memory.Affective biases were quantified using a choice test with the RAAD or vehicle administered 24 hours before testing to investigate the acute effects on retrieval.

Figure S7 :
Figure S7:Overview of the affective bias test protocol used to investigate the effects of RAADs on new learning.Animals were treated with either the RAAD or vehicle prior to each of the independent substrate-reward association learning sessions with any arising affective bias quantified using a choice test 24 hours after the last pairing session.

Figure S8 :
Figure S8:Overview of the affective bias test protocol used for the cue reactivation study.The protocol was similar to the method used to investigate the sustained effects of RAADs on a negative affective bias but included a cue-reactivation step on day 5, 1 hour after administration of ketamine (1mg/kg).Animals were treated with either FG7142 (3mg/kg) or corticosterone (10mg/kg) to induce a negatively biased memory and then re-exposed to either the FG7142-paired cue or the vehicle paired cue for 3 secs before being returned to their home cage.Affective biases were quantified using a choice test 24 hours after ketamine and cue-reactivation.

Figure S11 :
Figure S11: Cannula placements for animals used in the medial prefrontal cortex infusions.All animals' placements were verified post mortem and their data included in the analysis.

Figure S12 :
Figure S12: Turning the glass from half empty to half full: interactions between the acute pharmacological effects of RAADs, affective biases, and experience-dependent learning and memory which could explain their rapid and sustained effects on mood.We propose that ketamine, psilocybin and scopolamine act in mPFC to alter glutamate signalling and shift E/I balance generating a 'therapeutic window' where emotional circuits are selectively disengaged or reset to their default mode (PHASE I).During the arising 'therapeutic window', retrieval of memories can occur in the absence of their associated affective bias and, under appropriate conditions, can be re-activated and re-learnt with a relatively more positive affective bias (PHASE II).These findings and this arising hypothesis may provide the missing link between preclinical studies suggesting neuroplastic effects and the rapid and sustained improvements in mood observed in the clinic.

Table S1 :
List of the substrates used in the experiments in both cohorts.

Table S2A :
Standard procedure for testing drug-induced affective bias versus vehicle.Each animal receives drug treatment or vehicle counterbalanced over the four substrate-reward pairing sessions.Substrate (reward-paired substrates -'A' or 'B' versus unrewarded substrate -'C') and day are also counter-balanced resulting in four different groups.

Table S3 :
Summary of drug treatments in all cohorts.

Table S4 :
Pairing sessions data: number of trials to criterion and latency to dig in the rapid antidepressant effects studies.Data shown as mean (n=11-16 animals/group) ± SEM averaged from the two pairing sessions for each substrate-reward association (vehicle or drug).There were no significant effects during pairing sessions, either on response latency to dig or number of trials to criterion following treatment with vehicle or any of the drugs.

Table S5 :
Choice bias data: response latency to make choice in the rapid antidepressant effects studies.Data shown as mean (N=11-16 animals/group) ± SEM of an individual latencies during 30 trials of the choice test.No significant difference in latency to make choice was observed in studies following

Table S6 .
Choice test data: number of head twitches and wet dog shakes following vehicle and drug treatments in the rapid antidepressant effect study utilising psilocybin 0.1mg/kg and 0.3mg/kg.All drugs were administered 60 min.prior to the choice test.Animals were observed for 15 min.during testing session and the number of head twitches and wet dog shakes were scored.

Table S7 .
Choice test data: number of head twitches and wet dog shakes following vehicle and drug treatments in the rapid antidepressant effect study utilising psilocybin 1.0mg/kg.All drugs were administered 60 min.prior to the choice test.Animals were observed for 15 min.during testing session and the number of head twitches and wet dog shakes were scored.